Estrogens play key roles in the normal development and function of reproductive organs, mammary glands, bone, heart, vasculature, adipose, and central nervous system, as well as common dysfunctions of the same tissues. The long-term objective of these studies is to achieve a better understanding of the mechanisms by which liganded ERs control global patterns of gene expression to regulate cellular physiology. Our overall hypothesis is that biological context will play a key role in determining the nature and extent of estrogen-dependent transcriptional responses, and that estrogen responses will differ from the responses to other signaling pathways. In this proposal, we will use a unique, sensitive, powerful, and comprehensive new genomic approach for transcriptional profiling called Global nuclear Run-On and Sequencing (GRO-seq), which is superbly suited to the analysis of specific transcriptional mechanisms that cannot be addressed by other means. In the studies described herein, we will use GRO-seq in combination with other genomic, computational, and gene-specific assays, to elucidate the molecular and genomic mechanisms underlying the immediate and primary effects (i.e., minutes, not hours) of estrogen on the transcriptomes of normal and cancerous cells, including effects on non-coding transcripts. Specifically, we will: (1) determine the functional relationships between estrogen signaling pathways, ERa binding events, and estrogen-dependent transcriptional outcomes, (2) determine the mechanisms of regulation and functional roles of non-coding estrogen-regulated transcripts, with an emphasis on antisense transcripts, and (3) define and analyze the immediate/primary estrogen-regulated transcriptome in a variety of biological systems. These studies will exploit the power of GRO-seq to address fundamental molecular mechanisms and will reveal new mechanistic insights that will change the way we think about estrogen signaling. The increased understanding of estrogen signaling gained from these studies will aid in finding better ways to prevent, diagnose, and treat estrogen-related diseases. In addition, since ERs represent prototypical signal-regulated transcription factors, the study of ER function using GRO-seq will shed light on gene regulation by other transcription factors and suggest new ways to study their biology.